Fig. 4

Microglia preconditioned by crosstalk with metabolically stressed neuronal/glial co-cultures respond to a subsequent inflammatory stimulus. a Experimental timeline. Neuronal/glial co-cultures were exposed to metabolic stress, namely OGD or aglycemia for 30 min and then allowed to recover in regular culture medium for 24 h. Next, their supernatant (conditioned medium, CM) was used for further experiments. Untreated neuronal/glial co-cultures served as controls. The CM of all conditions was incubated with microglia for 20 h, then—as a strong subsequent inflammatory stimulus—LPS was applied at 10 ng/ml for 4 h. After this time, cells were used for further experiments. Microglia treated with and without LPS incubated in the same cell culture medium as the CM served as controls. b Representative immunocytochemical images of microglia incubated with the CM of neuronal/glial co-cultures. Iba1+ microglia (red) were co-stained with a nuclear marker (Hoechst; blue) in either untreated microglia (upper left panel), untreated microglia with LPS (upper middle panel), microglia with unstressed-CM and LPS (lower right panel) or microglia with OGD-CM with LPS (lower middle panel), or aglycemia-CM with LPS (lower right panel; scale bars = 50 μm), showing no morphological changes in the different conditions. c An IGF1-ELISA was used to quantify IGF1-release in the cell supernatant photometrically. There were no significant changes in IGF1 release between controls with and without LPS and microglia incubated with unstressed-CM + LPS, OGD-CM + LPS, or aglycemia-CM+ LPS (values displayed as means ± SEM of three independent experiments with n = 12/each condition; one-way ANOVA). d Q-PCR revealed no significant differences for the phagocytosis marker CD68 between cells treated with LPS only and cells treated with unstressed-CM + LPS, OGD-CM + LPS, and aglycemia-CM + LPS (one-way ANOVA). Each Q-PCR sample was normalized to RPL13a as the reference gene, and mRNA levels were normalized to endogenous RPL13a expression. e Q-PCR revealed that incubation with unstressed-CM + LPS increased the expression of the markers iNOS, IL-6, IL-1β, and IL-10 compared to cells treated with LPS only. Treatment with OGD-CM and aglycemia-CM, however, decreased the expression of iNOS, IL-6, IL-1β (only aglycemia-CM), and IL-10 (only OGD-CM; values displayed as means ± SEM of one representative experiment of three independent experiments with at least n = 12/each condition; the difference between CM-treated groups compared to control with LPS: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; the difference between OGD-CM and aglycemia-CM groups when compared to unstressed-CM group: ^#p < 0.05, ^##p < 0.01, ^###p < 0.001; one-way ANOVA, Tukey’s multiple comparison test). Each Q-PCR sample was normalized to RPL13a as the reference gene, and mRNA levels were normalized to endogenous RPL13a expression